Glucagon-like insulinotropic peptides, compositions and methods

ABSTRACT

The present invention provides novel complexes consisting of certain GLP-1 molecules associated with a divalent metal cation that is capable of co-precipitating with a GLP-1 molecule. Pharmaceutical compositions and methods of using such complexes for enhancing the expression of insulin in B-type islet cells is claimed, as is a method for treating maturity onset diabetes mellitus in mammals, particularly humans.

[0001] This application is a continuation-in-part of Galloway et al.,U.S. Ser. No. 08/164,277, filed Dec. 9, 1993.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the field of pharmaceutical and organicchemistry and provides novel compounds, and pharmaceutical compositionsthereof, which are useful for enhancing the expression of insulin frommammalian pancreatic B-type islet cells and for treating maturity onsetdiabetes mellitus in a mammal.

[0003] The endocrine secretions of the pancreatic islets are undercomplex control not only by blood-borne metabolites (glucose, aminoacids, catecholamines, etc.), but also by local paracrine influences.The major pancreatic islet hormones (glucagon, insulin and somatostatin)interact amongst their specific cell types (A, B, and D cells,respectively) to modulate secretory responses mediated by theaforementioned metabolites. Although insulin secretion is predominantlycontrolled by blood levels of glucose, somatostatin inhibitsglucose-mediated insulin secretory responses. In addition to theproposed interislet paracrine regulation of insulin secretion, there isevidence to support the existence of insulinotropic factors in theintestine. This concept originates from the observations that glucosetaken orally is a much more potent stimulant of insulin secretion thanis a comparable amount of glucose given intravenously.

[0004] The human hormone glucagon is a 29-amino acid peptide hormoneproduced in the A-cells of the pancreas. The hormone belongs to amulti-gene family of structurally related peptides that includesecretin, gastric inhibitory peptide, vasoactive intestinal peptide andglicentin. These peptides variously regulate carbohydrate metabolism,gastrointestinal mobility and secretory processing. The principalrecognized actions of pancreatic glucagon, however, are to promotehepatic glycogenolysis and glyconeogenesis, resulting in an elevation ofblood sugar levels. In this regard, the actions of glucagon are counterregulatory to those of insulin and may contribute to the hyperglycemiathat accompanies Diabetes mellitus [(Lund, P. K., et al., Proc. Natl.Acad. Sci. U.S.A., 79:345-349 (1982)].

[0005] Glucagon has been found to be capable of binding to specificreceptors which lie on the surface of insulin producing cells. Glucagon,when bound to these receptors, stimulates the rapid synthesis of cAMP bythese cells. cAMP, in turn, has been found to stimulate insulinexpression [Korman, L. Y., et al., Diabetes, 34:717-722 (1985)]. Insulinacts to inhibit glucagon synthesis [Ganong, W. F., Review of MedicalPhysiology, Lange Publications, Los Altos, Calif., p. 273 (1979)]. Thus,the expression of glucagon is carefully regulated by insulin, andultimately by the serum glucose level.

[0006] The glucagon gene is initially translated from a 360 base pairprecursor to form the polypeptide, preproglucagon [Lund, et al., Proc.Natl. Acad. Sci. U.S.A. 79:345-349 (1982)]. This polypeptide issubsequently processed to form proglucagon. Patzelt, C., et al., Nature,282:260-266 (1979), demonstrated that proglucagon was subsequentlycleaved into glucagon and a second polypeptide. Subsequent work by Lund,P. K., et al., Lopez L. C., et al., Proc. Natl. Acad. Sci. U.S.A.,80:5485-5489 (1983), and Bell, G. I., et al., Nature 302:716-718 (1983),demonstrated that the proglucagon molecule was cleaved immediately afterlysine-arginine dipeptide residues. Studies of proglucagon produced bychannel catfish (Ictalurus punctata) indicated that glucagon from thisanimal was also proteolytically cleaved after adjacent lysine-argininedipeptide residues [Andrews P. C., et al., J. Biol. Chem., 260:3910-3914(1985), Lopez, L. C., et al., Proc. Natl. Acad. Sci. U.S.A.,80:5485-5489 (1983)]. Bell, G. I., et al., supra, discovered thatmammalian proglucagon was cleaved at lysine-arginine orarginine-arginine dipeptides, and demonstrated that the proglucagonmolecule contained three discrete and highly homologous peptidemolecules which were designated glucagon, glucagon-like peptide 1(GLP-1) and glucagon-like peptide 2 (GLP-2). Lopez, et al., concludedthat glucagon-like peptide 1 was 37 amino acid residues long and thatglucagon-like peptide 2 was 34 amino acid residues long. Analogousstudies on the structure of rat preproglucagon revealed a similarpattern of proteolytic cleavage between adjacent lysine-arginine orarginine-arginine dipeptide residues, resulting in the formation ofglucagon, GLP-1 and GLP-2 [Heinrich, G., et al., Endocrinol.,115:2176-2181 (1984)]. Human, rat, bovine, and hamster sequences ofGLP-1 have been found to be identical [Ghiglione, M., et al.,Diabetologia, 27:599-600 (1984)].

[0007] The conclusion reached by Lopez, et al., regarding the size ofGLP-1 was confirmed by the work of Uttenthal, L. O., et al., J. Clin.Endocrinol. Metabol., 61:472-479 (1985). Uttenthal, et al., examined themolecular forms of GLP-1 which were present in the human pancreas. Theirresearch shows that GLP-1 and GLP-2 are present in the pancreas as 37amino acid and 34 amino acid peptides, respectively.

[0008] The similarity between GLP-1 and glucagon suggested to earlyinvestigators that GLP-1 might have biological activity. Although someinvestigators found that GLP-1 could induce rat brain cells tosynthesize cAMP [Hoosein, N. M., et al., Febs Lett. 178:83-86 (1984)],other investigators failed to identify any physiological role for GLP-1(Lopez, L. C., et al.). The failure to identify any physiological rolefor GLP-1 caused some investigators to question whether GLP-1 was infact a hormone and whether the relatedness between glucagon and GLP-1might be artifactual.

[0009] Variants of GLP-1 (7-37) and analogs thereof, also have beendisclosed. These variants and analogs include, for example, Gln⁹-GLP-1(7-37), D-Gln⁹-GLP-1 (7-37), acetyl-Lys⁹-GLP-1 (7-37), Thr⁶-Lys¹⁸-GLP-1(7-37), Lys¹⁸-GLP-1 (7-37) and the like, and derivatives thereofincluding, for example, acid addition salts, carboxylate salts, loweralkyl esters, and amides [see, e.g., WO 91/11457]. Generally, thevarious disclosed forms of GLP-1 are known to stimulate insulinsecretion (insulinotropic action) and cAMP formation [see, e g., Mojsov,S., Int. J. Peptide Protein Research, 40:333-343 (1992)].

[0010] More importantly, multiple authors have demonstrated the nexusbetween laboratory experimentation and mammalian, particularly human,insulinotropic responses to exogenous administration of GLP-1,particularly GLP-1 (7-36) NH₂ and GLP-1 (7-37) [see, e.g., Nauck, M. A.,et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., NewEngland J. of Medicine, 326(20):1316-1322 (1992); Nauck, M. A., et al.,J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes,42:1219-1225 (1993)].

[0011] More particularly, the fundamental defects identified as causinghyperglycemia in maturity onset diabetes are impaired secretion ofendogenous insulin and resistance to the effects of insulin by muscleand liver [Galloway, J. S., Diabetes Care, 13:1209-1239, (1990)]. Thelatter defect results in excessive production of glucose from the liver.Thus, whereas a normal individual releases glucose at the rate ofapproximately 2 mg/kg/minute, in patients with maturity onset diabetes,this amount usually exceeds 2.5 mg/kg/minute resulting in a net excessof at least 70 grams of glucose per 24 hours. The fact that there existsexceedingly high correlations between hepatic glucose production, thefasting blood glucose and overall metabolic control as indicated byglycohemoglobin measurements [Galloway, J. A., supra; and Galloway, J.A., et al., Clin. Therap., 12:460-472 (1990)], it is readily apparentthat control of the fasting blood glucose is a sine quo non forachieving overall normalization of metabolism sufficient to prevent thecomplication of hyperglycemia. In view of the fact that present forms ofinsulin rarely normalize hepatic glucose production without producingsignificant hyperinsulinemia and hypoglycemia (Galloway, J. A., andGalloway, J. A., et al., supra) alternative approaches are needed.

[0012] Intravenous infusions of GLIP-1 (7-36)NH₂ to produce twice normalserum concentrations have been demonstrated to produce the effectsindicated in the table below: Patients With Normal Maturity OnsetSubjects Diabetes Meal glycemia (1) Unchanged Reduced Fasting glycemia(2) — Reduced Fasting glucagon (2) — Reduced Post-prandial glucagon (1)— Reduced Endogenous insulin Unchanged Increased secretion in responseto a meal (1) Free fatty acids Reduced (3) Reduced (2)

[0013] However, the long-term stability of GLP-1, particularly GLP-1 asa component of a pharmaceutical composition for administration tomammals, is questionable. In fact, when stored at the low temperature of4° C., by-products of GLP-1 (7-37) have been found as early as elevenmonths after sample preparation (Mojsov, S., supra). Thus, there existsa need for a more stable GLP-1 compound which can safely be administeredto mammals in need of such treatment.

[0014] Furthermore, the biological half-life of GLP-1 molecules,particularly those molecules which are affected by the activity ofdipeptidyl-peptidase IV (DPP IV) is quite short. For example, thebiological half-life of GLP-1 (7-37) is a mere 3 to 5 minutes (U.S. Pat.No. 5,118,666), and is further influenced by its rapid absorptionfollowing parenteral administration to a mammal. Thus, there also existsa need for a GLP-1 compound which delays absorption following suchadministration.

[0015] Accordingly, the present invention provides compounds whichsatisfy the aforementioned stability requirements. The compounds of thepresent invention also provide delayed absorption following parenteraladministration and, consequently, should have extended biologicalhalf-lives. Also provided are pharmaceutical compositions of thecompounds of the present invention, as well as methods for using suchcompounds.

SUMMARY OF THE INVENTION

[0016] The present invention provides a complex consisting of a divalentmetal cation associated with and capable of co-precipitating with acompound of the formula:

R₁-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂

[0017] wherein:

[0018] R₁ is selected from the group consisting of L-histidine,D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;

[0019] X is selected from the group consisting of Ala, Gly, Val, Thr,Ile, and alpha-methyl-Ala;

[0020] Y is selected from the group consisting of Glu, Gln, Ala, Thr,Ser, and Gly;

[0021] Z is selected from the group consisting of Glu, Gln, Ala, Thr,Ser, and Gly;

[0022] R₂ is selected from the group consisting of NH₂, and Gly-OH;providing that the compound has an isoelectric point in the range fromabout 6.0 to about 9.0 and further providing that when R₁ is His, X isAla, Y is Glu, and Z is Glu, R₂ must be NH₂.

[0023] Also provided by the present invention is a pharmaceuticalcomposition comprising a compound of the present invention incombination with one or more pharmaceutically acceptable carriers,diluents, or excipients.

[0024] The present invention further provides a method for enhancing theexpression of insulin comprising providing to a mammalian pancreaticB-type islet cell an effective amount of a compound of the presentinvention, as well as a method of treating maturity onset diabetesmellitus which comprises administering to a mammal in need of suchtreatment an effective amount of a compound of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025] One aspect of the present invention provides a complex consistingof a GLP-1 molecule having an isoelectric point in the range from about6.0 to about 9.0, complexed with a divalent metal cation.

[0026] As used in the present specification, the term “GLP-1 moleculesrefer” to naturally-occurring GLP-1 (7-36)NH₂, GLP-1 (7-37), natural andunnatural functional analogs and derivatives thereof, and salts thereof.The amino acid sequence of GLP-1 (7-36)NH₂ is well known in the art, butis presented below as a convenience to the reader:

His⁷-Ala-Glu-Gly¹⁰-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu²⁰-Glu-Gly-Gln-Ala-Ala²⁵-Lys-Glu-Phe-Ile-Ala³⁰-Trp-Leu-Val-Lys-Gly³⁵-Arg-NH₂.

[0027] For GLP-1 (7-37), the carboxy-terminal amide functionality ofArg³⁶ is displaced with Gly at the 37th position of the GLP-1 (7-36)NH₂molecule.

[0028] In addition, the existence and preparation of a multitude ofprotected, unprotected, and partially protected natural and unnaturalfunctional analogs and derivatives of GLP-1 (7-36)NH₂ and GLP-1 (7-37)molecules have been described in the art [see, e.g., U.S. Pat. Nos.5,120,712 and 5,118,666, which are herein incorporated by reference, andOrskov, C., et al., J. Biol. Chem., 264(22):12826-12829 (1989) and WO91/11457 (Buckley, D. I., et al., published Aug. 8, 1991)].

[0029] As known in the art, amino acid residues may be in theirprotected form in which both amino and carboxy groups possessappropriate protecting groups, partially-protected form in which eitheramino or carboxy groups possess appropriate protecting groups, orunprotected form in which neither amino nor carboxy groups possess anappropriate protecting group. Numerous reactions for the formation andremoval of such protecting groups are described in a number of standardworks including, for example, “Protective Groups in Organic Chemistry”,Plenum Press (London and New York, 1973); Green, T. H., “ProtectiveGroups in Organic Synthesis”, Wiley (New York, 1981); and “ThePeptides”, Vol. I, Schröder and Lübke, Academic Press (London and NewYork, 1965).

[0030] Representative amino protecting groups include, for example,formyl, acetyl, isopropyl, butoxycarbonyl, fluorenylmethoxycarbonyl,carbobenzyloxy, and the like.

[0031] Representative carboxy protecting groups include, for example,benzyl ester, methyl ester, ethyl ester, t-butyl ester, p-nitro phenylester, and the like.

[0032] In addition to protected forms in which both amino and carboxygroups possess appropriate protecting groups, the term “protected” alsorefers to those GLP-1 molecules in which the activity ofdipeptidyl-peptidase IV is resisted or inhibited [see, e.g., Mentlein,R., et al., Eur. J. Biochem., 214:829-835 (1993)]. In addition toGLP-1(7-36) NH₂, molecules which are protected from the activity of DPPIV are preferred, and Gly⁸-GLP-1(7-36)NH₂, Val⁸-GLP-1(7-37) OH,α-methly-Ala⁸-GLP-1(7-36)NH₂, and Gly⁸-Gln²¹-GLP-1(7-37)OH are morepreferred.

[0033] Derivatives of naturally-occurring GLP-1 molecules are thosepeptides which are obtained by fragmenting a naturally-occurringsequence, or are synthesized based upon a knowledge of the sequence ofthe naturally-occurring amino acid sequence of the genetic material (DNAor RNA) which encodes this sequence. The term “derivatives” alsoincludes chemical modification of natural or unnatural GLP-1 molecules.Processes for preparing these derivatives are well known to organic andpeptide chemists of ordinary skill (see, e.g., WO 91/11457, supra).

[0034] GLP-1 molecules of the present invention also include analogs ofGLP-1 (7-36)NH₂ and GLP-1 (7-37) in which one or more amino acids whichare not present in the original sequence are added or deleted, andderivatives thereof. Specifically, His and desamino-histidine arepreferred for R₁, so long as the overall isoelectric point of themolecule is in the range of about 6 to 9. Ala, Gly, and Val arepreferred at the “X” position, so long as the overall isoelectric pointof the mnolecule is in the range of about 6 to 9. Likewise, Glu, and Glnare preferred at the “Y” position, so long as the overall isoelectricpoint of the molecule is in the range of about 6 to 9. Also, Glu, andGln are preferred at the “Z” position, so long as the overallisoelectric point of the molecule is in the range of about 6 to 9.Finally, Gly-OH is preferred for R₂, so long as the overall isoelectricpoint of the molecule is in the range of about 6 to 9.

[0035] Furthermore, the present invention includes a salt form of aGLP-1 molecule. A GLP-1 of this invention can possess a sufficientlyacidic, a sufficiently basic, or both functional groups, and accordinglyreact with any of a number of inorganic bases, and inorganic and organicacids, to form a salt. Acids commonly employed to form acid additionsalts are inorganic acids such as hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, phosphoric acid, and the like, andorganic acids such as p-toluenesulfonic acid, methanesulfonic acid,oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid,citric acid, benzoic acid, acetic acid, and the like. Examples of suchsalts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like. Preferred acid addition salts are those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, and,especially, hydrochloric acid.

[0036] Base addition salts include those derived from inorganic bases,such as ammonium or alkali or alkaline earth metal hydroxides,carbonates, bicarbonates, and the like. Such bases useful in preparingthe salts of this invention thus include sodium hydroxide, potassiumhydroxide, ammonium hydroxide, potassium carbonate, and the like. Thesalt forms are particularly preferred.

[0037] Of course, when the compounds of this invention are used forpharmacotherapeutic purposes, those compounds may also be in the form ofa salt, but the salt must be pharmaceutically acceptable.

[0038] Thus, GLP-1 molecules of the present invention include interalia, those GLP-1 molecules which functionally demonstrateinsulinotropic activity. The term “insulinotropic activity” relates tothe ability of a substance to stimulate, or cause the stimulation of,the synthesis or expression of the hormone insulin.

[0039] The insulinotropic property of a compound may be determined byproviding that compound to animal cells, or injecting that compound intoanimals and monitoring the release of immunoreactive insulin (IRI) intothe media or circulatory system of the animal, respectively. Thepresence of IRI is detected through the use of a radioimmunoassay whichcan specifically detect insulin.

[0040] Although any radioimmunoassay capable of detecting the presenceof IRI may be employed, it is preferable to use a modification of theassay method of Albano, J. D. M., et al., Acta Endocrinol., 70:487-509(1972). In this modification, a phosphate/albumin buffer with a pH of7.4 is employed. The incubation is prepared with the consecutiveaddition of 500 μl of phosphate buffer, 50 μl of perfusate sample or ratinsulin standard in perfusate, 100 μl of anti-insulin antiserum(Wellcome Laboratories; 1:40,000 dilution), and 100 μl of [¹²⁵I)insulin, giving a total volume of 750 μl in a 10×75 mm disposable glasstube. After incubation for 2-3 days at 4° C., free insulin is separatedfrom antibody-bound insulin by charcoal separation. The assaysensitivity is 1-2 uU/mL. In order to measure the release of IRI intothe cell culture medium of cells grown in tissue culture, one preferablyincorporates radioactive label into proinsulin. Although any radioactivelabel capable of labelling a polypeptide can be used, it is preferableto use ³H leucine in order to obtain labelled proinsulin. Labelling canbe done for any period of time sufficient to permit the formation of adetectably labelled pool of proinsulin molecules; however, it ispreferable to incubate cells in the presence of radioactive label for a60 minute time period.

[0041] Although many cell lines capable of expressing insulin can beused for determining whether a compound has an insulinotropic effect, itis preferable to use rat insulinoma cells, and especially RIN-38 ratinsulinoma cells. Such cells can be grown in any suitable medium;however, it is preferable to use DME medium containing 0.1% BSA and 25mM glucose.

[0042] The insulinotropic property of a compound may also be determinedby pancreatic infusion. The in situ isolated perfused rat pancreaspreparation is a modification of the method of Penhos, J. C., et al.,Diabetes, 18:733-738 (1969). Fasted male Charles River strain albinorats, weighing 350-600 g, are anesthetized with an intraperitonealinjection of Amytal Sodium (Eli Lilly and Co.: 160 ng/kg). Renal,adrenal, gastric, and lower colonic blood vessels are ligated. Theentire intestine is resected except for about four cm of duodenum andthe descending colon and rectum. Therefore, only a small part of theintestine is perfused, minimizing possible interference by entericsubstances with glucagon-like immunoreactivity. The perfusate is amodified Krebs-Ringer bicarbonate buffer with 4% dextran T70 and 0.2%bovine serum albumin (fraction V), and is bubbled with 95% O₂ and 5%CO₂. A nonpulsatile flow, 4-channel roller bearing pump (Buchlerpolystatic, Buchler Instruments Division, Nuclear-Chicago Corp.) isused, and a switch from one perfusate source to another is accomplishedby switching a 3-way stopcock. The manner in which perfusion isperformed, monitored, and analyzed follow the method of Weir, G. C., etal., J. Clin. Inestigat. 54:1403-1412 (1974), which is herebyincorporated by reference.

[0043] The GLP-1 molecules of the present invention are required topossess a histidine functionality at the amino terminus. GLP-1 moleculesof the present invention may also possess a modified histidinefunctionality in lieu of the required histidine functionality.

[0044] The term “modified histidine” refers to a histidine functionalitywhich has been chemically or biologically altered or an alteredhistidine functionality which has been synthesized de novo, but whichretains its metal binding properties.

[0045] Numerous such modified histidine functionalities and theirpreparation are known in the art and include, for example, D-histidine(WO 91/11457), desamino-histidine (WO 92/18531), 2-amino-histidine[Levine-Pinto, H., et al., Biochem. Biophys. Res. Commun.,103(4):1121-1130 (1981)], β-hydroxy-L-histidine [Owa, T, et al.,Chemistry Letters, pp. 1873-1874 (1988)], L-homohistidine [Altman, J.,et al., Synthetic Commun., 19(11&12):2069-2076 (1989)],α-fluoromethyl-histidine (U.S. Pat. NO. 4,347,374), andα-methylhistidine [O'Donnell, M. J., Synthetic Commun.,19(7&8):1157-1165 (1989)].

[0046] The GLP-1 molecules of the present invention further are requiredto have an isoelectric point in the range from about 6.0 to about 9.0.Numerous GLP-1 molecules having an isoelectric point in this range havebeen disclosed and include, for example:

[0047] GLP-1 (7-36)NH₂

[0048] Gly⁸-GLP-1 (7-36)NH₂

[0049] Gln⁹-GLP-1 (7-37)

[0050] D-Gln⁹-GLP-1 (7-37)

[0051] acetyl-Lys⁹-GLP-1 (7-37)

[0052] Thr⁹-GLP-1 (7-37)

[0053] D-Thr⁹-GLP-1 (7-37)

[0054] Asn⁹-GLP-1 (7-37)

[0055] D-Asn⁹-GLP-1 (7-37)

[0056] Ser²²-Arg²³-Arg²⁴-Gln²⁶-GLP-1 (7-37)

[0057] Thr¹⁶-Lys¹⁸-GLP-1 (7-37)

[0058] Lys¹⁸-GLP-l (7-37)

[0059] Arg²³-GLP-1 (7-37)

[0060] Arg²⁴-GLP-1 (7-37), and the like (see, e.g., WO 91/11457, supra)In addition, GLP-1 molecules of the present invention, when possessingeach of the above-referenced modified histidine functionalities in lieuof the histidine functionality, have isoelectric points which fallwithin the above-defined range. Methods for calculating orexperimentally determining the isoelectric point of other GLP-1molecules are known to one of ordinary skill in the art.

[0061] Methods for preparing the GLP-1 molecules of the presentinvention also are well known to an ordinarily skilled peptide chemist.

[0062] In one method, GLP-1 molecules are prepared by the well-knownsolid phase peptide synthetic schemes described by Merrifield, J. M.,Chem. Soc., 85:2149 (1962), and Stewart and Young, Solid Phase PeptideSynthesis, pp. 24-66, Freeman (San Francisco, 1969). However, it also ispossible to obtain fragments of the proglucagon polypeptide or of GLP-1(1-37) by fragmenting the naturally-occurring amino acid sequence using,for example, a proteolytic enzyme. Further, it is possible to obtain thedesired fragments of the proglucagon peptide or of GLP-1 (1-37) throughthe use of recombinant DNA technology as disclosed by Maniatis, T., etal., Molecular Biology: A Laboratory Manual, CSH (Cold Spring Harbor,1982).

[0063] Likewise, the state of the art in molecular biology provides theordinarily skilled artisan another means by which compounds of thepresent invention can be obtained. Although it may be produced by solidphase peptide synthesis or recombinant methods, recombinant methods maybe preferable because higher yields are possible. The basic steps inrecombinant production are:

[0064] a) isolating a natural DNA sequence encoding a GLP-1 molecule orconstructing a synthetic or semi-synthetic DNA coding sequence for aGLP-1 molecule,

[0065] b) placing the coding sequence into an expression vector in amanner suitable for expressing proteins either alone or as a fusionproteins,

[0066] c) transforming an appropriate eukaryotic or prokaryotic hostcell with the expression vector,

[0067] d) culturing the transformed host cell under conditions that willpermit expression of a GLP-1 molecule, and

[0068] e) recovering and purifying the recombinantly produced GLP-1molecule.

[0069] As previously stated, the coding sequences may be whollysynthetic or the result of modifications to the larger, nativeglucagon-encoding DNA. A DNA sequence that encodes preproglucagon ispresented in Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349(1982) and may be used as starting material in the semisyntheticproduction of the compounds of the present invention by altering thenative sequence to achieve the desired results.

[0070] Synthetic genes, the in vitro or in vivo transcription andtranslation of which results in the production of a GLP-1 molecule, maybe constructed by techniques well known in the art. Owing to the naturaldegeneracy of the genetic code, the skilled artisan will recognize thata sizable yet definite number of DNA sequences may be constructed, allof which encode GLP-1 molecules.

[0071] The methodology of synthetic gene construction is well known inthe art. See Brown, et al. (1979) Methods in Enzymology, Academic Press,N.Y., Vol. 68, pgs. 109-151. DNA sequences that encode a GLP-1 moleculecan be designed based on the amino acid sequences herein disclosed. Oncedesigned, the sequence itself may be generated using conventional DNAsynthesizing apparatus such as the Model 380A or 380B DNA synthesizers(PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City,Calif. 94404).

[0072] To effect expression of a GLP-1 molecule, one inserts theengineered synthetic DNA sequence in any one of many appropriaterecombinant DNA expression vectors through the use of appropriaterestriction endonucleases. See generally Maniatis et al. (1989)Molecular Cloning; A Laboratory Manual, Cold Springs Harbor LaboratoryPress, N.Y., Vol. 1-3. Restriction endonuclease cleavage sites areengineered into either end of the GLP-1 molecule-encoding DNA tofacilitate isolation from, and integration into, known amplification andexpression vectors. The particular endonucleases employed will bedictated by the restriction endonuclease cleavage pattern of the parentexpression vector to be employed. The choice of restriction sites arechosen so as to properly orient the coding sequence with controlsequences to achieve proper in-frame reading and expression of theprotein of interest. The coding sequence must be positioned so as to bein proper reading frame with the promoter and ribosome binding site ofthe expression vector, both of which are functional in the host cell inwhich the protein is to be expressed.

[0073] To achieve efficient transcription of the synthetic gene, it mustbe operably associated with a promoter-operator region. Therefore, thepromoter-operator region of the synthetic gene is placed in the samesequential orientation with respect to the ATG start codon of thesynthetic gene.

[0074] A variety of expression vectors useful for transformingprokaryotic and eukaryotic cells are well known in the art. See ThePromega Biological Research Products Catalogue (1992) (Promega Corp.,2800 Woods Hollow Road, Madison, Wis., 53711-5399); and The StratageneCloning Systems Catalogue (1992) (Stratagene Corp., 11011 North TorreyPines Road, La Jolla, Calif., 92037). Also, U.S. Pat. No. 4,710,473describes circular DNA plasmid transformation vectors useful forexpression of exogenous genes in E. coli at high levels. These plasmidsare useful as transformation vectors in recombinant DNA procedures and

[0075] (a) confer on the plasmid the capacity for autonomous replicationin a host cell;

[0076] (b) control autonomous plasmid replication in relation to thetemperature at which host cell cultures are maintained;

[0077] (c) stabilize maintenance of the plasmid in host cellpopulations;

[0078] (d) direct synthesis of a protein prod. indicative of plasmidmaintenance in a host cell population;

[0079] (e) provide in series restriction endonuclease recognition sitesunique to the plasmid; and

[0080] (f) terminate mRNA transcription.

[0081] These circular DNA plasmids are useful as vectors in recombinantDNA procedures for securing high levels of expression of exogenousgenes.

[0082] Having constructed an expression vector for a GLP-1 molecule, thenext step is to place the vector into a suitable cell and therebyconstruct a recombinant host cell useful for expressing the polypeptide.Techniques for transforming cells with recombinant DNA vectors are wellknown in the art and may be found in such general references asManiatis, et al. supra. Host cells made be constructed from eithereukaryotic or prokaryotic cells.

[0083] Prokaryotic host cells generally produce the protein at higherrates and are easier to culture. Proteins which are expressed inhigh-level bacterial expression systems characteristically aggregate ingranules or inclusion bodies which contain high levels of theoverexpressed protein. Such protein aggregates typically must besolubilized, denatured and refolded using techniques well known in theart. See Kreuger, et al. (1990) in Protein Folding, Gierasch and King,eds., pgs 136-142, American Association for the Advancement of SciencePublication No. 89-18S, Washington, D.C.; and U.S. Pat. No. 4,923,967.

[0084] Once the desired GLP-1 molecule is prepared, providing it has anisoelectric point in the range from about 6.0 to about 9.0, complexes ofthe present invention are prepared by complexing a desired GLP-1molecule with a divalent metal cation via well known methods in the art.Such metal cations include, for example, Zn⁺⁺, Mn⁺⁺, Fe⁺⁺, Co⁺⁺, Cd⁺⁺,Ni⁺⁺, and the like. Of the metal cations, Zn⁺⁺ is preferred.

[0085] Generally, a desired GLP-1 molecule, having the requiredisoelectric point, is combined with a mixture of an appropriate bufferand an appropriate form of a metal cation.

[0086] Appropriate buffers are those which will maintain the mixture ata pH range from about 6.0 to about 9.0, but which will not interferewith the reaction. Preferred buffers include Goode's buffers,particularly HEPES, and Tris and Tris acetate.

[0087] Appropriate forms of metal cations are any form of a divalentmetal cation which is available to form a complex with a GLP-1 moleculeof the present invention. Preferably, a divalent metal cationic saltsuch as zinc chloride is provided in excess to provide a molar ratio ofup to about 50 molecules of a divalent metal cation for each molecule ofGLP-1 substrate.

[0088] The temperature employed in this step is that which is sufficientto effect completion of the reaction. Typically, the reaction is run atambient temperature.

[0089] The product of the present reaction, a crystalline or amorphoussuspension, is isolated and purified using standard techniques.

[0090] The present invention also provides pharmaceutical compositionscomprising a compound of the present invention in combination with apharmaceutically acceptable carrier, diluent, or excipient. Suchpharmaceutical compositions are prepared in a manner well known in thepharmaceutical art, and are administered individually or in combinationwith other therapeutic agents, preferably via parenteral routes.Especially preferred routes include intramuscular and subcutaneousadministration.

[0091] Parenteral daily dosages, preferably a single, daily dose, are inthe range from about 1 pg/kg to about 1,000 μg/kg of body weight,although lower or higher dosages may be administered. The requireddosage will depend upon the severity of the condition of the patient andupon such criteria as the patient's height, weight, sex, age, andmedical history.

[0092] In making the compositions of the present invention, the activeingredient, which comprises at least one compound of the presentinvention, is usually mixed with an excipient or diluted by anexcipient. When an excipient is used as a diluent, it may be a solid,semi-solid, or liquid material which acts as a vehicle, carrier, ormedium for the active ingredient.

[0093] In preparing a formulation, it may be necessary to mill theactive compound to provide the appropriate particle size prior tocombining with the other ingredients. If the active compound issubstantially insoluble, it ordinarily is milled to particle size ofless than about 200 mesh. If the active compound is substantially watersoluble, the particle size is normally adjusted by milling to provide asubstantially uniform distribution in the formulation, e.g., about 40mesh.

[0094] Some examples of suitable excipients include lactose, dextrose,sucrose, trehalose, sorbitol, and mannitol. The compositions of theinvention can be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after administration to the patient byemploying procedures well known in the art.

[0095] The compositions are preferably formulated in a unit dosage formwith each dosage normally containing from about 50 μg to about 100 mg,more usually from about 1 mg to about 10 mg of the active ingredient.The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with a suitablepharmaceutical excipient.

[0096] For the purpose of parenteral administration, compositionscontaining a compound of the present invention preferably are combinedwith distilled water and the pH is adjusted to about 6.0 to about 9.0.

[0097] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb a compound of the presentinvention. The controlled delivery may be exercised by selectingappropriate macromolecules (for example, polyesters, polyamino acids,polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose,carboxymethylcellulose, and protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release.

[0098] Another possible method to control the duration of action bycontrolled release preparations is to incorporate a compound of thepresent invention into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers.

[0099] Alternatively, instead of incorporating a compound into thesepolymeric particles, it is possible to entrap a compound of the presentinvention in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules, respectively, or incolloidal drug delivery systems, for example, liposomes, albuminmicrospheres, microemulsions, nanoparticles, and nanocapsules, or inmacroemulsions. Such teachings are disclosed in Remington'sPharmaceutical Sciences (1980).

[0100] The compounds of the present invention have insulinotropicactivity. Thus, another aspect of the present invention provides amethod for enhancing the expression of insulin comprising providing to amammalian pancreatic B-type islet cell an effective amount of a compoundof the present invention.

[0101] Similarly, the present invention provides a method for treatingmaturity onset diabetes mellitus in a mammal, preferably a human, inneed of such treatment comprising administering an effective amount of acompound or composition of the present invention, to such a mammal.

[0102] The following examples are provided to further illustrate thepresent invention. It is not intended that the invention be limited inscope by reason of any of the following examples.

EXAMPLE 1

[0103] Individual aliquots of 5 different GLP-1 molecules were preparedby well-known, solid phase peptide synthesis and were lyophilized insmall vials. Portions of 0.1M HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) buffers at pH7.4 containing various levels of zinc chloride were added to thealiquots to obtain a protein concentration of about 0.1 mg/mL. Thesamples were mixed and stored at ambient temperature (22° C.) for about18 hours. The mixtures were then centrifuged (Fisher Model 235Cmicro-centrifuge) for 5 minutes. The clear supernatants were pipettedfrom the tubes. The protein content of the supernatants was estimated bymeasuring their absorbance at 280 nm in a spectrophotometer (Gilford260). The theoretical absorbance value for a 0.1 mg/mL solution of theGLP-1 molecules at this wavelength in the 1 cm cuvettes is 0.207. Theresults of this experiment are shown in Table 1. TABLE 1 Zn/GLP-1Molecule 280 nm Absorbance α-methyl- Gly⁸-Gln²¹ Molar GLP-1 Gly⁸-GLP-1Val⁸-GLP-1 Ala⁸-GLP-1 GLP-1 Ratio (7-36)NH₂ (7-36)NH₂ (7-37)OH (7-36)NH₂(7-37)OH 0 0.172 0.136 0.187 0.163 0.167 0.3 0.099 0.079 0.191 0.1340.113 0.5 0.057 0.070 0.184 0.098 0.082 0.7 0.035 0.058 0.180 0.0790.069 1.0 0.039 0.057 0.173 0.076 0.065 3.0 0.048 0.044 0.110 0.0550.055

[0104] This example shows that only a small quantity of zinc is requiredto complex with and precipitate a significant portion of the GLP-1molecules from these dilute solutions.

EXAMPLE 2

[0105] 5 mg of GLP-1 (7-36)NH₂ was completely dissolved in 2.5 ML of pH7.4, zinc-free 0.1M HEPES buffer. An additional 2.5 mL of pH 7.4, 0.1MHEPES buffer containing 0.6 mM zinc chloride was quickly added. Theapproximate molar ratio of zinc to GLP-1 (7-36)NH₂ in this sample is 1to 1. The solution immediately became cloudy and precipitation soonformed. The mixture was stored at ambient temperature (22° C.) for 18hours.

[0106] The precipitate became firmly attached to the bottom of the glassvial. The supernatant was completely decanted by pipette. Theprecipitate was then completely dissolved in 5.0 mL of 0.1N hydrochloricacid. The absorbance at 280 nm was determined for both the supernatantand redissolved precipitate solutions. The zinc levels in thesesolutions were quantitated by atomic absorption spectrophotometry. Theresults of this experiment are shown in Table 2. TABLE 2 ZincConcentration 280 nm in Parts per Absorbance Million Supernatant (5 ml)0.118 9.02 Redissolved Precipitate (5 ml) 1.932 13.3

[0107] This example shows that most of the GLP-1 (7-36)NH₂ precipitatedfrom the solution when the zinc-containing HEPES solution was added. The280 nm absorbance value of 1.932 indicates the GLP-1 (7-36)NH₂concentration of the redissolved precipitate is 0.933 mg/ml, or 283 μM.The zinc concentration of this same solution, 13.3 parts per million, isequivalent to a zinc concentration of 203 μM. Therefore, the molar ratioof zinc to GLP-1 (7-36)NH₂ in the precipitate was 0.717-to 1.

1 1 1 30 PRT Homo sapiens MOD_RES (30)..(30) The arginine residue atposition 30 is modified so as to replace the terminal carboxyl groupwith an amine. 1 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr LeuGlu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys GlyArg 20 25 30

We claim:
 1. A complex consisting of a divalent metal cation associatedwith and capable of co-precipitating with a compound of the formula:R₁-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂wherein: R₁ is selected from the group consisting of L-histidine,D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, andalpha-methyl-Ala; Y is selected from the group consisting of Glu, Gln,Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu,Gln, Ala, Thr, Ser, and Gly; R₂ is selected from the group consisting ofNH₂, and Gly-OH; providing that the compound has an isoelectric point inthe range from about 6.0 to about 9.0 and further providing that when R₁is His, X is Ala, Y is Glu, and Z is Glu, R₂ must be NH₂.
 2. A complexof claim 1 wherein said divalent metal cation is zinc.
 3. A complex ofclaim 2 wherein R₁ is chosen from the group consisting of His anddesamino-histidine.
 4. A complex of claim 2 wherein X is chosen from thegroup consisting of Ala, Gly, and Val.
 5. A complex of claim 2 wherein Yis chosen from the group consisting of Glu and Gln.
 6. A complex ofclaim 2 wherein Z is chosen from the group consisting of Glu and Gln. 7.A complex of claim 2 wherein R₁ is His, X is Val, Y is Glu, Z is Glu,and R₂ is Gly-OH.
 8. A complex of claim 2 wherein R₁ is His, X is Gly, Yis Gln, Z is Glu, and R₂ is Gly-OH.
 9. A pharmaceutical compositionwhich comprises a complex according to claim 1 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.10. A pharmaceutical composition which comprises a complex according toclaim 2 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 11. A pharmaceutical compositionwhich comprises a complex according to claim 3 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.12. A pharmaceutical composition which comprises a complex according toclaim 4 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 13. A pharmaceutical compositionwhich comprises a complex according to claim 5 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.14. A pharmaceutical composition which comprises a complex according toclaim 6 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 15. A pharmaceutical compositionwhich comprises a complex according to claim 7 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.16. A pharmaceutical composition which comprises a complex according toclaim 8 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 17. A method of treating maturityonset diabetes mellitus which comprises administering to a mammal inneed of such treatment an effective amount of a complex according toclaim 1 to said mammal.
 18. A method of treating maturity onset diabetesmellitus which comprises administering to a human in need of suchtreatment an effective amount of a complex according to claim 1 to saidmammal.
 19. A method of treating maturity onset diabetes mellitus whichcomprises administering to a human in need of such treatment aneffective amount of a complex according to claim 2 to said mammal.
 20. Amethod of treating maturity onset diabetes mellitus which comprisesadministering to a human in need of such treatment an effective amountof a complex according to claim 3 to said mammal.
 21. A method oftreating maturity onset diabetes mellitus which comprises administeringto a human in need of such treatment an effective amount of a complexaccording to claim 4 to said mammal.
 22. A method of treating maturityonset diabetes mellitus which comprises administering to a human in needof such treatment an effective amount of a complex according to claim 5to said mammal.
 23. A method of treating maturity onset diabetesmellitus which comprises administering to a human in need of suchtreatment an effective amount of a complex according to claim 6 to saidmammal.
 24. A method of treating maturity onset diabetes mellitus whichcomprises administering to a human in need of such treatment aneffective amount of a complex according to claim 7 to said mammal.
 25. Amethod of treating maturity onset diabetes mellitus which comprisesadministering to a human in need of such treatment an effective amountof a complex according to claim 8 to said mammal.
 26. A method forenhancing the expression of insulin comprising providing to a mammalianpancreatic B-type islet cell an effective amount of a complex ofclaim
 1. 27. A process of preparing a GLP-1 complex, which comprisessteps of; a) mixing a GLP-1 molecule of the formula:R₁-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂ wherein: R₁ is selected from the group consisting of L-histidine,D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;X is selected from the group consisting of Gly, Val, Thr, Ile, andalpha-methyl-Ala; Y is selected from the group consisting of Glu, Gln,Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu,Gln, Ala, Thr, Ser, and Gly; R₂ is selected from the group consisting ofNH₂, and Gly-OH;  providing that the compound has an isoelectric pointin the range from about 6.0 to about 9.0;  with a divalent metal cationto form a mixture and; b) incubating for a time and a temperaturesufficient to form a complex.
 28. The process of claim 27, wherein X isselected from the group consisting of Val, Thr, Ile, andalpha-methyl-Ala.
 29. The process of claim 27, wherein R₁ isL-histidine, X is Val, Y is Glu, Z is Glu, and R₂ is Gly-OH.
 30. Theprocess of claim 27, wherein RI is L-histidine, X is Gly oralpha-methyl-Ala, Y is Glu, Z is Glu, and R₂ is —NH₂.
 31. The process ofclaim 27, wherein the divalent metal cation is zinc.
 32. The process ofclaim 28, wherein the divalent metal cation is zinc.
 33. The process ofclaim 29, wherein the divalent metal cation is zinc.
 34. The process ofclaim 30, wherein the divalent metal cation is zinc.
 35. The process ofclaim 27, further comprising the step of adding a buffer to the mixture.36. The process of claim 35 wherein the buffer has a pH in the range ofabout 6 to about
 9. 37. The process of claim 31, wherein the zinc isprovided at a molar ratio of up to about 50 molecules of zinc for eachmolecule of GLP-1.
 38. The process of claim 37, wherein the molar ratiois about 1 molecule of zinc for each molecule of GLP-1.
 39. The processof claim 37, wherein the molar ratio is about 0.7 molecules of zinc foreach molecule of GLP-1.